Anat Cell Biol.  2015 Mar;48(1):1-9. 10.5115/acb.2015.48.1.1.

When morphogenetic proteins encounter special extracellular matrix and cell-cell connections at the interface of the renal stem/progenitor cell niche

• Minuth WW
• Denk L

• KMID: 2046069
• DOI: http://doi.org/10.5115/acb.2015.48.1.1

Abstract

Reciprocal exchange of morphogenetic proteins between epithelial and mesenchymal cells in a stem/progenitor cell niche results in formation of a nephron. To maintain diffusion of morphogenetic proteins, it is assumed that a close contact exists between involved cells. However, recent publications underline that both types of stem/progenitor cells are separated by a striking interface. To explore this microarchitecture in detail, neonatal rabbit kidneys were fixed in traditional glutaraldehyde (GA) solution for transmission electron microscopy. For contrast enhancing specimens were fixed in GA solution including cupromeronic blue, ruthenium red or tannic acid. To record same perspectives, embedded blocks of parenchyma were cut in exactly orientated vertical and transverse planes to lining collecting ducts. Electron microscopy of specimens fixed by traditional GA solution illustrates a spatial separation of stem/progenitor cells and an unobstrusively looking interface. In contrast, advanced fixation of specimens in GA solution including cupromeronic blue, ruthenium red and tannic acid unmasks earlier not visible extracellular matrix. In addition, projections of mesenchymal cells covered by matrix cross the interface to contact epithelial cells. Surprisingly, the end of a mesenchymal cell projection does not dangle but is enclosed in a fitting sleeve and connected via tunneling nanotubes with the plasma membrane of an epithelial cell. Regarding this complex ensemble the question is to what extent illustrated cell-cell connections and extracellular matrix are involved in communication and transmission of morphogenetic proteins during induction of a nephron.

Keyword

Kidney; Development; Stem/progenitor cell niche; Cell contacts; Tunneling nanotubes

MeSH Terms

Cell Membrane
Diffusion
Epithelial Cells
Extracellular Matrix*
Glutaral
Kidney
Microscopy, Electron
Microscopy, Electron, Transmission
Nanotubes
Nephrons
Ruthenium Red
Strikes, Employee
Tannins
Glutaral
Ruthenium Red
Tannins

Figure

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  Fig. 1 (A-E) Vertical (B-D) and transversal (E) views to the renal stem/progenitor cell niche. A paraffin section of fetal human (B) and a semithin section of neonatal rabbit kidney (C) demonstrate an ureteric bud derived collecting duct (CD) ampulla (A) enclosing epithelial cells. Mesenchymal cells are separated by a gap (asterisks). A niche is covered by an organ capsule (C), while at the lateral sides a renal vesicle and a forming S-shaped body are recognized. (D) Schematic illustration shows vertical sections 1 to 4 of the niche. (E) In a semithin section the different planes exhibit that epithelial cells are separated from mesenchymal cells by a gap (asterisks). Human kidney (gestational age between week 16 and 18) was obtained from the stock of preparations used for the Course of Microscopic Anatomy for medical students at the University of Regensburg, Germany.

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  Fig. 2 (A-E) Transversal view to the stem/progenitor cell niche of neonatal rabbit kidney by transmission electron microscopy. Specimens were fixed in traditional glutaraldehyde (GA) solution (B) or GA solution including cupromeronic blue (CMB) (C), ruthenium red (RR) (D), or tannic acid (TA) (E). Epithelial cells enclosed in a collecting duct ampulla (A) are separated from mesenchymal cells by a gap (asterisks). (E) Fixation in GA solution including TA elucidates that in the gap special extracellular matrix is contained.

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  Fig. 3 (A-E) Analysis of the interface within the stem/progenitor cell niche of neonatal rabbit kidney by transmission electron microscopy. Specimens were fixed by traditional glutaraldehyde (GA) solution (B) and GA solution including cupromeronic blue (CMB) (C), ruthenium red (RR) (D), or tannic acid (TA) (E). It can be seen that the distance between mesenchymal and epithelial cells is constant so that both are separated by a striking interface (asterisks). Further projections (P) of mesenchymal cells cross the interface to contact the basal lamina of epithelial cells enclosed within a collecting duct (CD) ampulla. (B) Fixation by GA solution informs that the interface (asterisk) looks bright. (C) Fixation by GA solution including CMB points out that crossing cell projections and the basal lamina of epithelial cells are covered by a punctuate pattern of proteoglycans (arrowhead). (D) Fixation by GA solution including RR unveils a band of extracellular matrix covering the basal lamina of epithelial cells. Cell projections of mesenchymal cells are partially wrapped by this dense label. (E) Fixation by GA solution including TA shows intense label on the basal lamina of epithelial cells. Mesenchymal cell projections are covered by TA label. The basal lamina at the tip of a CD ampulla consists of a lamina rara (L.r.), lamina densa (L.d.) and lamina fibroreticularis (L.f.). The basal plasma membrane of an enclosed epithelial cell is marked by a cross (+).

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  Fig. 4 (A-I) Analysis of mesenchymal cell projections contacting epithelial cells within the stem/progenitor cell niche of neonatal rabbit kidney by transmission electron microscopy. Mes, mesenchymal cell; E, epithelial cell. Specimens were fixed by traditional glutaraldehyde (GA) solution (B, F) and GA solution including cupromeronic blue (CMB) (C, G), ruthenium red (RR) (D, H), or tannic acid (TA) (E, I). (B-E) A mesenchymal cell projection (P) is crossing the interface (asterisks) to contact the basal lamina of epithelial cells. (B) Fixation in GA solution shows that the end of a projection has a tender contact on the basal lamina via microfibers. (C) Fixation in GA solution including CMB informs that a cell projection is mounted by proteoglycan braces (arrowhead). (D) Fixation in GA solution including RR elucidates that a projection is covered by a sleeve of filigree extracellular matrix. (E) Fixation in GA solution including TA demonstrates that a projection is fastened by a labeled sleeve. (D, E) In the center of a projection (marked area) tunneling nanotubes (arrows) are present connecting mesenchymal with epithelial cells. Traditional fixation does not (F), but improved fixation elucidates in a transversal perspective that a mesenchymal cell projection does not dangle but is enclosed by extracellular matrix (G-I). The basal lamina at the tip of a collecting duct ampulla consists of a lamina rara (L.r.), lamina densa (L.d.) and lamina fibroreticularis (L.f.). The basal plasma membrane of enclosed epithelial cell is marked by a cross (+).

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  Fig. 5 (A-M) Analysis of the contact between the end of a mesenchymal cell projection and an epithelial cell within the stem/progenitor cell niche of neonatal rabbit kidney by transmission electron microscopy (B-I) and schematic illustration (J-M). Mes, mesenchymal cell; E, epithelial cell; P, projection. Specimens were fixed by traditional glutaraldehyde (GA) solution (B, F) and GA solution including cupromeronic blue (CMB) (C, G), ruthenium red (RR) (D, H), or tannic acid (TA) (E, I). In a vertical perspective it is recognized that the end of a mesenchymal cell projection is crossing the interface (asterisk) to have a special contact on the basal lamina of epithelial cells. The distance between the end of a projection and the plasma membrane of an epithelial cell is in average 167 nm. (B) Fixation in GA solution shows that the end of a projection has a contact on the basal lamina via tiny microfibers. (C) Fixation in GA solution including CMB illustrates that a cell projection is mounted in a cylinder of proteoglycan braces (arrowhead). (D) Fixation in GA solution including RR shows that a projection is integrated in a sleeve of extracellular matrix. (E) Fixation in GA solution including TA depicts that a projection is enveloped in labeled extracellular matrix. (F-I, J-M) In the center of a projection tunneling nanotubes (arrows) are seen connecting mesenchymal with epithelial cells. The basal lamina at the tip of a collecting duct ampulla consists of a lamina rara (L.r.), lamina densa (L.d.) and lamina fibroreticularis (L.f.). The basal plasma membrane of enclosed epithelial cell is marked by a cross (+).

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